PROJECT SUMMARY Since the discovery of combined anti-retroviral therapy (cART) the incidence of many of the complications of infection with HIV-1 have been dramatically decreased. One exception to this trend are neurologic complications. HIV associated neurocognitive disorders (HAND) is a spectrum of disorders that can range from mild cognitive problems to dementia. While the incidence of dementia has decreased, the milder forms of HAND has remained basically unchanged despite our ability to reduce circulating virus to undetectable levels. The exact cause of HAND largely remains a mystery. Virus quickly invades the brain and is thought to establish a foothold in microglia and infiltrating macrophages. After initiation of therapy even though the amount of virus decreases significantly, levels of some viral proteins persist and are thought to contribute to immune activation. One such protein is Nef. Even cells that are completely virally suppressed can continue to produce Nef. We have shown that production of Nef is sufficient to drive the production of exosomes. Indeed, it has been shown that astrocytes, transfected with the gene for Nef and placed into healthy brains of Rats can induce neurocognitive defects. These data suggest that both Nef and astrocytes play a key role in the pathogenesis of HAND. We hypothesize that infected astrocytes produce exosomes, which can interact with other cells in the brain and upregulate the expression and secretion of amyloid to cause cognitive impairment. In this proposal, we will determine whether Nef exosomes are present in the plasma and CSF of HAND patients with mild cognitive impairment (ANI or MND), determine if their numbers correlate with severity of disease, and determine if these exosomes can induce amyloid expression and markers of neuronal inflammation using a primary iPSC brain model (mini-brains). To determine if CSF exosomes are derived from astrocytes, we will use magnetic beads with antibody attached to the astrocyte-specific marker GLAST. We will also determine if human primary astrocytes, infected by HIV-1, can produce Nef exosomes and could be source of CSF exosomes. This study will provide important information on the role that Nef exosomes could play in the development of HAND and provide important insight into this increasingly important complication of HIV/AIDS.